This invention relates to nucleotide sequences coding for a human protein having angiogenesis regulative properties.
More particularly, this invention relates to the isolation and to the molecular characterization of a gene coding for a new protein having the properties of an angiogenic factor which regulates in vivo the formation and/or the regeneration of the vertebrate blood vessel system, and it also relates to the protein itself. Moreover, this invention also refers to vectors containing such sequence or parts thereof, to prokaryotic and eukaryotic cells transformed with such vectors, and to the employment of such vectors and of such cells for the production of the protein and of corresponding polyclonal and/or monoclonal antibodies as well.
It is well known that growth factors are polypeptides, synthesized and secreted by mammalian cells, capable of acting not only on the proliferation, but also on the differentiation and morphogenesis of target cells. Indeed, it has been shown that some growth factors exert their action by regulating mechanisms such as chemotaxis, activation of inflammatory system cells and repairing of tissues (Whitman, M. and Melton, D. A., 1989, Annual Rev. Cell Biol., 5. 93-117).
Because of the similar phenotype between cultured growth factors stimulated and retrovirus transformed cells, it has been suggested that common mechanisms control such phenomena. Indeed, the interaction between a growth factor and its own specific receptor indirectly activates gene activity regulative proteins, through intermediate reactions involving different protein-kinases. Many of the components of this metabolic chain have been identified as the cellular analogs of viral oncogenes, suggesting how oncoviruses could interfere with normal cellular processes.
Many growth factors have been identified up to the present time, the corresponding genes have been cloned, and such factors have been divided into groups, on the basis of similar activities and/or of sequence homologies; among them there is the family of angiogenic factors.
Angiogenesis, or the formation of vessels of the vascular system, is a complex process occuring during embroyogenesis, wound healing and organ regeneration. Moreover, some pathologies like the growth of solid tumors, some retinopathies and rheumatoid arthritis induce an aberrant angiogenesis (Risau W., 1990, Progress in Growth Factor Research, 2, 71-79).
Angiogenesis in vivo is a multi-step process, two of them being represented by the migration and the proliferation of endothelial cells devoted to the formation of vessels.
In the most recent years, many angiogenic factors have been identified, and the corresponding genes cloned. Among them: angiogenin, subject-matter of the patent application PCT no.8701372; the platelet-derived endothelial growth factor PD-ECGF (Ishigawa et al., 1989, Nature, 338, 557); the human vascular permeability factor, VPF (Keck et al., 1989, Science 246, 1309), which was cloned also in the mouse with the denomination of vascular endothelial growth factor, VEGF (Leung et al., 1989, Science, 246, 1306); the growth factor for fibroblasts, i.e., the acid factor, a-FGF, and the basis factor, b-FGF, the transforming growth factors alpha, TGF-.alpha., and beta, TGF-.beta.(Folkman and Klagsburn, 1987, Science, 235, 442).
Angiogenic factors have been divided into two groups, according to their way of action: either directly on the vascular endothelial cells, by stimulating motility or mitosis, or indirectly on cells producing growth factor acting on endothelial cells.
In vitro analysis have put into evidence that angiogenic factors exert different effects on the motility and on the proliferation of endothelial cells. Indeed, some of them stimulate just one of the two events, other ones stimulate both events, whereas others seem to be ineffective in vitro, and, lastly, other ones show even an inhibiting activity of the endothelial cellular proliferation. Such data point out that the regulation of angiogenesis is a complex process mediated by different components, many of which have not been identified as yet.
Accordingly it is evident that there is the need for identifying and isolating new angiogenic factors capable of stimulating the migration and differentiation of endothelial cells, to be utilized both in the diagnostic field, as tumoral markers and for inflammatory diseases, and in the therapeutical field, for topic or internal use, for instance in the treatment of wounds, of tissues after a surgical operation, of transplantation, of burns, ulcers, etc.. Such factors can be employed successfully also in vitro, as growth-stimulating of cell cultures.
Moreover, DNA recombinant techniques allow such factors to be produced in suitable amounts, in short times and at remarkably low costs.
Indeed, there is an increasing need for identifying new specific tumoral markers because of uncertainties in tumor diagnosis. Moreover, recent methods for producing hybrid proteins (Fitzgerald D. and Pastan I., 1989, J. Natl. Cancer Inst. 81, 1455-1463) and/or conjugate antibodies (Pearson, J. W. et al., 1989 Cancer Res. 49, 3562-3567) with toxic molecules, are giving promising results in the field of tumoral serotherapy, with an increasingly growing demand for new factors to test. Finally, many of angiogenic factor genes have been cloned starting from tumoral cells, whereas a better applicability in the therapeutic field of genes coming from non-neoplastic material is evident.